In vitro anti-leishmanial and anti-trypanosomal activity of hydrazones,

pyrazoles, pyrazolo[1,5-a]pyrimidines and pyrazolo[3,4-b]pyridine,

synthesized from 6-substituted-3-formylchromones

Elier Galarraga1,*, Neudo Urdaneta 1, and Julio C. Herrera1

1 Chemistry department, Simón Bolívar University , 89000 Caracas 1080A,

Venezuela.

* Corresponding author: eliergalarraga@usb.ve

1

Graphical Abstract

In vitro anti-leishmanial and anti-trypanosomal activity of hydrazones,

pyrazoles, pyrazolo[1,5-a]pyrimidines and pyrazolo[3,4-b]pyridine,

synthesized from 6-substituted-3-formylchromones

2

Abstract

Led by the biological and pharmacological relevance of the 3-

formylchromone derivatives and its interesting chemistry, in this work we present the

synthesis of a series of pyrazoles (4a-c), hydrazones (5a-c), pyrazolo[1,5-

a]pyrimidines (6a, 6b) and one pyrazolo[3,4-b]pyridine (7) and the report on their in

vitro anti-leishmanial and anti-trypanosomal activity. Chemical results showed that the

formation of regioisomer 7 may arise from an imine intermediary that undergoes 1,4-

addition by attack of C-4' from the pyrazole. To the best of our knowledge, this is the

first report regarding formation of pyrazolo[3,4-b]-pyridines by intramolecular attack of

an sp2 carbon atom.

The in vitro studies were performed against strains of Leishmania mexicana

(bel 21) and Tripanosoma cruzi (DM28). Compounds 5a and 5b showed activity at

micromolar level and good selectivity index (SI) with IC50 values of 6.3 (SI = 3.4) and

15 (SI = 1.9) mM for L. Mexicana and 4.1 (SI = 5.2) and 10 (SI = 3) mM for T. cruzi

respectively. From the above-mentioned, compounds 5a and 5b may be considered

for further chemical modifications in order to increase their activity as potential

antiparasitic agents.

Keywords: Anti-leishmanial, anti-trypanosomal, 6-substituted-3-formylchromones

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Introduction

The research of compounds with antiparasitic properties is a matter of great

importance. Chaga’s disease is present mostly in countries of South America with about 5-6

million individuals infected and 25 million at risk [1]. Leishmaniasis on the other hand, is a

growing public health problem, with an annual incidence of about 1.3 million cases [2].

Nitrogenated derivatives such as hydrazones, pyrazoles, pyrazolo[1,5-a]pyrimidines

and pyrazolo[3,4-b]pyridines are widely recognized as an important class of biological active

substances [3-8]. They have shown several biological and pharmacological activities, including

cytotoxic, antitrichomonal, antischistosomal, antichagasic and antimalarial effects. The

presence of these nitrogenated groups is also observed in commercial drugs. Mebendazole

[9], a well known antiparasitic compound, presents amino imidazole and methanone moieties

while the antileishmaial drug allopurinol [10] bears a pyrazolo[1,5-a]-pyrimidine nucleus.

In a previous work, high toxicities of pyrazole 4a-c hydrazones 5a-c pyrazolo[1,5-

a]pyrimidines 6a, 6b and pyrazolo [3,4-b]pyridine 7 compounds were observed against

Artemia salina through the brine shrimp lethality assay (BSLA) [11]. This anti-larvae property

may be indicative of trypanocidal action [12]. In this study we decided to evaluate the in vitro

antileishmanial and antitrypanosomal activity of those nitrogenated derivatives, against strains

of L. mexicana and T. cruzi.

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Results and discussion

Synthesis

The products 4a-c, 5a-c, 6a and 6b were obtained from the reaction between

equimolar quantities of 3-formylchromone derivatives 1a-c, with the corresponding hydrazines

2a, 2b or aromatic amine 3 in anhydrous THF (Scheme 1).

The pyrazolo[3,4-b]pyridine 7 was obtained in two steps: the reaction of 1b with 3

produced a precipitate which was then refluxed using AcOH as solvent in presence of I2

(Scheme 1). The formation of regioisomer 8 may arise from an imine intermediary (Scheme 2)

that undergoes 1,4-addition at C-2 by attack of C-4' from the pyrazole instead of the nitrogen

atom N-2'. To the best of our knowledge, this is the first report regarding formation of

pyrazolo[3,4-b]-pyridines by intramolecular attack of an sp2 carbon atom.

All reactions were monitored by TLC and obtained in good yields (80–95%). The

compounds were fully characterized using NMR, IR and MS methods; all physical constants and

spectroscopic data were in accordance with those reported in the literature [11,13,14,19].

5

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Scheme 1. Reagents and conditions: (i) THF, reflux

(2h); (ii) AcOH/I2, reflux (4h)

Scheme 2. Formation of pyrazolo[3,4-b]pyridine 7

Antileishmanial and anti-trypanosomal activity

The nitrogenated compounds 4a-c, 5a-c, 6a, 6b and 7 were tested for leishmanicidal and

trypanocidal activities. Primary cultures of human dermal fibroblast were used as reference. The

selectivity action of the compound over the parasite was calculated using the selectivity index

(SI), which is expressed as the ratio of IC50 on fibroblast to the IC50 of the corresponding

parasite strain.

As observed in Table 1, all tested compound showed good to moderate activity.

Compound 5a exhibited the strongest antiparasitic activity against both parasites L. mexicana

and T. cruzi with IC50 values of 6.3 ± 0.7 M and 4.1 ± 0.3 M respectively. Compound 5a also

displayed the highest SI with values of 3.4 for L. mexicana and 5.2 for T. cruzi. Therefore, 5a

was the most effective compound tested against both parasites. Compounds 5b and 5c also

exhibited good antiparasitic activity since their IC50 values were relatively low. When 5b was

tested against L. Mexicana, the IC50 was 15 ± 3 M having a SI value of 1.9, while against T.

cruzi the IC50 value was 10 ± 1 M with a SI value of 3. 5c showed activity only for T.cruzi (IC50 =

13 ± 1 M) with SI value of 2. Compounds 4b and 7 exhibited moderate activity against L.

mexicana (IC50 = 25 ± 2 and 35 ± 2 M, respectively) but in the case of compound 7 the

selectivity was poor.

7

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We compared the antiparasitic results action of 4a-c, 5a-c, 6a-6b and 7 with their

antilarvae properties [11], and found that the most active compounds against L. mexicana

and T. cruzi were also among the most toxic to A. salina. We also evaluated the correlation

between leishmanicidal and trypanocidal activities of the synthesized compounds, using a

linear regression method. There was a significant association (p<0.05) between both

antiparasitic effects. The correlation coefficient was 0.71, indicating a moderately strong

relationship. So the most active compounds could be evaluated in a wider range of parasite

strains.

The strongest antiparasitic activity was found in the series 5a-c, which has a

hydrazone-type structure bearing a 2,4-dinitrophenyl moiety. The hydrazone nucleus has

been found to be important in the leishmanicidal activity [15]. The presence of the nitro

groups could also contribute to the activity since they are able to play a role in antiprotozoal

substances [16-18].

From the above-mentioned, compounds 5a and 5b may be considered for further

chemical modifications, in order to increase their activity and selectivity as potential

antiparasitic agents.

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Table 1: In vitro leishmanicidal and trypanocidal activities of synthesized compounds

against L. mexicana and T. cruzi.

IC50: concentration of the compound that induces 50% growth inhibition in 48 h. Values are expressed as the mean ± SD; SI:

selectivity index, is defined as the ratio of IC50 on fibroblasts to the IC50 on the corresponding parasite; ND: not determinated.

Comp. L. mexicana T. cruzi Fibroblasts

IC50 (M) SI IC50 (M) SI IC50 (M)

4a 37 ± 4 0.3 57 ± 9 <0.7 11 ± 1

4b 25 ± 2 3.0 > 90 <0.8 73 ± 11

4c 53 ± 13 - 174 ± 60 - ND

5a 6.3 ± 0.7 3.4 4.1 ± 0.3 5.2 22 ± 5

5b 15 ± 3 1.9 10 ± 1 3.0 28 ± 7

5c > 23 1.3 13 ± 1 2.2 29 ± 4

6a 89 ± 9 - 107 ± 25 - ND

6b 60 ± 3 - 93 ± 16 - ND

7 35 ± 2 0.6 35 ± 4 0.6 19.5 ± 0.4

Materials & Methods

General procedure for the synthesis of 4a-c and 5a-c

3-Formylchromone derivative 1a-c (1.0 mmol) was dissolved in hot anhydrous THF (4

mL) and a solution of hydrazines 2a, 2b (1.0 mmol) in THF (2 mL) was added slowly. The

mixture was refluxed for 1–2 h, and once cooled the solid was filtered, washed with water and

recrystallized from absolute EtOH.

General procedure for the synthesis of 6a and 6b

3-Formylchromone derivative 1a or 1c (1.0 mmol) was mixed with aminopyrazole 3

(1.0 mmol) and refluxed in 10 mL of anhydrous THF for 1 h. After cooling, the solid was filtered,

washed repeatedly with hot THF and recrystallized from EtOH to give TLC pure compounds.

General procedure for the synthesis of 7

Equimolar quantities of 1b and 3 (1.0 mmol) were refluxed in anhydrous THF for 1-2

h, until the formation of a precipitate. Once separated from the solution, this precipitate was

dissolved in AcOH (7 mL) in presence of I2 (1.0 mmol) and refluxed for a period of 3-4 h. After

completion of the reaction by TLC, the mixture was poured into crushed ice and treated with

NaHCO3 and Na2SO3. The solid was filtered, washed with cold water and dried.

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Antileishmanial and antitrypanosomal activity

Epimastigotes of T. cruzi and promastigotes of L. mexicana were grown at 26°C in

liver infusion tryptose (LIT) medium with penicillin (100 U/mL), streptomycin (100 µg/mL), and

10% heat-inactivated FBS. A modified MTT assay was used to determine the compounds IC50

on each parasite strain [20]. Briefly, 199 μL per well of exponential growing parasites at a

dilution of 2×106 cells per mL, were trasfered into a 96-well flat-bottom plate, and compounds or

solvents (1 µL compound or DMSO) were added to evaluate different concentrations. Plates

were incubated at 26°C for 48h and 5 days for L. mexicana promastigotes or T. cruzi

epimastigotes respectively. Then, parasites were seeded, the medium was removed, and the

cells were incubated with MTT solution in PBS (0.2 mg/ml for L. mexicana or 0.5 mg/ml for T.

cruzi), F-formazan crystals were dissolved by addition of 100 µL DMSO after plates were

centrifuged and MTT solution was removed. The following equation was used to calculate the

percentage of growth inhibition (%GI):

%GI = [1 – (Ap – Ab)/ Ac – Ab)] × 100

Ap is the absorbance of compound-treated cells, Ac is the absorbance of solvent-

treated cells, and Ab is the absorbance of culture medium. The IC50 value was calculated

applying a sigmoidal analysis when %GI and the corresponding concentration were plotted

using the Origin 8.0® software (OriginLab Corporation). For each condition evaluated at least

three independent assays were performed in triplicate.

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Conclusions

All synthesized compounds exhibited good to moderate antiparasitic activities

against strains of L. mexicana and T. cruzi.

Compounds 5a and 5b showed the strongest activities with good selectivity

indexes. The results suggest that these molecules are suitable candidates for

further chemical modification and evaluation as potential anti-leishmanial and anti-

trypanosomal agents.

The good correlation exhibited for the results between both parasite lines

indicates that the most active compounds 5a and 5b, could be used in a wider

range of parasite strains.

Finally, the comparison of the antiparasitic results with the previous BSLA study

allowed us to conclude that this anti-larvae test may be a good and inexpensive

tool in predicting trypanocidal action.

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Acknowledgments

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